This invention relates to an apparatus for freezing fertilized ova, spermatozoa or the like.
Artificial fertilization has been recently frequently carried out to contrive the improvements of breed of domestic animal and of growth of the domestic animal. In this case, fertilized ova and spermatozoa have been preserved by freezing the same.
It is heretofore known to fill the fertilized ova and spermatozoa in a buffer solution contained in a tube and to freeze the buffer solution as a method of freezing the fertilized ova and spermatozoa.
A temperature change with respect to time of a pure substance when the substance is cooled under a constant pressure is generally known as a cooling curve. According to this principle, the substance does not always start to immediately freeze when the substance reaches its freezing point, but the substance will start generally freezing after the substance is overcooled to the temperature lower than its freezing point. Simultaneously, the substance raises its temperature to its true freezing point, and the substance will then lower its temperature again after the entire substance is completely frozen.
The buffer solution is overcooled at the freezing time in the freezing step according to the foregoing conventional freezing method which merely cools the substance. Then, the temperature of the substance or buffer solution is thereafter immediately raised. Therefore, the fertilized ova and spermatozoa are disadvantageously killed due to the thermal shock of this abrupt temperature change according to the conventional freezing method.
It has been proposed to avoid such a thermal shock in a method of freezing fertilized ova and spermatozoa to remove the buffer solution cooled to the freezing point and to hold a tube containing the buffer solution with a pincette preserved in liquefied nitrogen, thereby removing the freezing from the holding portion. This also lacks actual utilization due to the fact that the removal of the tube adversely affects the temperature of the buffer solution to fail in the freezing of the solution, so as and an automatic control is difficult due to the complicated operations.
Then, the present applicant has proposed a novel method, as disclosed in Japanese patent application No. 124,996/1981, of freezing fertilized ova, spermatozoa or the like comprising the steps of irregularly containing articles to be frozen such as fertilized ova, spermotozoa or the like in a buffer solution in a tube, cooling a buffer solution without the articles to be frozen with predetermined refrigerant so that the buffer solution without the articles to be frozen reaches a lower temperature than another buffer solution containing the articles to be frozen, thereby producing crystalline nuclei by freezing the buffer solution without articles to be frozen, and then cooling the crystalline nuclei so that the nuclei grow to the buffer solution containing the articles to be frozen to freeze the buffer solution containing the articles to be frozen, thereby freezing the article to be frozen such as fertilized ova, spermatozoa or the like.
More particularly, in this method, a buffer solution 2 is prepared by dissolving, for example, dimethyl sulfoxide (DMSO), dextrose, glycerin and/or sodium citrate in distilled water, the buffer solution 2 thus prepared is filled in a tube 1, e.g., a straw tube as shown in FIG. 1 or 2, and articles 3 to be frozen, e.g., fertilized ova, spermatozoa or the like are thrown into the buffer solution 2.
In this case, a cotton plug 4 or the like is charged in the lower end of the tube 1, and the articles 3 to be frozen are, on the other hand, floated in the middle of the buffer solution 2, or in this case the article 3 to be frozen is contained an irregular position at the lower half portion of the buffer solution 2, in which case segmenting gaps 5 may sometimes be formed with air bubbles at a certain position or positions or the buffer solution 2 as shown in FIG. 2.
When the above tube 1 is then cooled, the tube 1 is not cooled, but the difference of phases is provided at the cooling temperature between a buffer solution segment 2a containing the fertilized ova, spermatozoa or the like as the articles 3 to be frozen in the middle portion of the tube 1 and a buffer solution segment 2b containing no article 3 in the lower portion of the tube 1, the buffer solution segment 2b is first frozen to form crystalline nuclei, and the crystalline nuclei thus produced are then grown to the buffer solution segment 2a, thereby freezing the articles 3, e.g., the fertilized ova, spermatozoa or the like.